Project Summary: Maintaining blood-brain barrier (BBB) integrity is essential for a healthy central nervous system. Many pathological conditions, including HIV infection, brain trauma, and alcohol use disorders (AUD), are known to compromise the integrity of the BBB. Binge drinking of hard liquor [>40% alcohol by volume (ABV)] is a popular activity among adolescents and young adults. Our preliminary studies indicate that binge drinking of alcoholic beverages with a high ethanol (EtOH) concentration can cause severe BBB damage. Thus, excessive alcohol consumption poses an increased risk for those individuals who are already susceptible to BBB dysfunction. Brain mircrovascular endothelial cells (BMVECs) are key cellular components of the BBB. BMVECs contain tight junction (TJ) complexes, consisting of occludin, claudins, and zonula occludens (ZO), that restrict paracellular passage of substances across the BBB and ensure the structural and functional integrity of the BBB. The death (apoptosis) of BMVECs is a biomarker of BBB damage, and can be caused by alcohol abuse. However, the mechanisms by which alcohol affects the recovery of BBB integrity are not fully understood, but may include alcohol-delayed repair of the BMVEC lining. While studies have shown that apoptosis of BMVECs can cause BBB damage, there has been little, if any, investigation into the repair of the BMVEC lining and the restoration of BBB integrity. This is due, mainly, to the lack of an appropriate experimental animal model in which BBB integrity is already compromised. Intermedilysin (ILY), a toxin secreted by Streptococcus intermedius, binds exclusively to the species- specific human cell membrane protein, CD59 (hCD59). We previously demonstrated that injection of ILY into a transgenic mouse strain that expresses hCD59 on endothelial cells induces rapid endothelial cell damage in a dose-dependent manner. We recently established a Cre-inducible floxedSTOP-hCD59 transgenic mouse line (ihCD59) and crossed the inducible hCD59 with Tek-cre to generate compound mice (ihCD59+/-/Tek-cre+/- (+/-, hemizygous for the transgene) in which Cre expression drives the expression of hCD59 specifically in endothelial cells. Thus, we can generate an endothelial cell-specific ablation model using ILY administration. Endothelial progenitor cells (EPCs) can differentiate into endothelial cells and repair an injured endothelial cell lining. Based on the literature and the data from our preliminary studies, we hypothesize that (a) alcohol- induced damage to the endothelial lining of an already compromised BBB is EtOH concentration- dependent; and (b) repair and restoration of the damaged endothelial cell lining is delayed by binge alcohol consumption. To test this hypothesis, we propose to utilize our novel ILY-mediated cell ablation technique to generate an already compromised BBB model. Then, we will examine EtOH concentration- dependent effects on the damaged BBB in that model, including the repair and restoration of the BBB endothelial cell lining. The two Specific Aims of this proposal are: Aim 1: To determine the mechanisms underlying damage to the endothelial cell lining of the BBB following binge exposure to various concentrations of EtOH. Aim 2: To determine the mechanisms underlying the repair of the endothelial cell lining of the BBB following binge exposure to various concentrations of EtOH. The findings from this highly significant and clinically relevant study will provide valuable information on the mechanisms by which alcohol abuse further damages an already compromised BBB, and the timeframe of repair and restoration of BBB integrity.